CN101691330A - Separation and purification methods of highly purified antiviral active components in artichoke - Google Patents
Separation and purification methods of highly purified antiviral active components in artichoke Download PDFInfo
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Abstract
The invention discloses a method for efficiently separating and preparing highly purified antiviral active components (single caffeoylquinic acid compound and dicaffeoylquinic acid compound) in artichoke, mainly comprising the following steps: utilizing alcohol aqueous solution to extract fresh or dry artichoke, concentrating, carrying out column chromatographic separation, preparing crude extract rich in the antiviral active components, carrying out separation and purification on the crude extract by combing high speed counter-current chromatography with high preparative performance liquid chromatography, and finally adopting a recrystallization method for refining to obtain highly purified 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoyl quinine acid and 4,5-di-O-caffeoylquinic acid. The method is suitable for preparing highly purified monomer by utilizing various natural products or the extractives of the natural products containing one or more components containing the above caffeoylquinic acid compounds as raw materials, wherein the natural products or the extractives are obtained from various ways; and the method has simple steps, simple operation, high efficiency, low cost and large separating preparation quantity, is easy to repeat; and the purification of the caffeoylquinic acid compounds prepared by the method can be up to 98%.
Description
Technical field
The present invention relates to the separation purification method of effective constituent in Chinese medicine or the natural product, relate generally to the method for extraction, separating monomer compound from Chinese medicine or natural product, be specifically related to a kind of high purity coffee mesitoyl quinine acid compounds 1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, the 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4, the method for separating and preparing of 5-two-O-caffeoylquinic acids.
Background technology
Arithoke (Cynara scolymus L. has another name called cynara scolymus, choke, foreign lily, French lily, lotus lily) is the composite family per nnial herb.Originate in the Mediterranean Sea bank.China the nineties in 20th century from overseas introduction, at present all there is large-scale plantation on ground such as Hunan, Zhejiang and Yunnan.The bud of arithoke (meat holder and phyllary), cauline leaf be medicine-food two-purpose from ancient times, has abundant edible and physiologically active and is worth, and tradition is used for maldigestion, hepatobiliary disease, diuresis detoxifcation, lipopenicillinase, step-down etc.Pharmacological research shows, contains multiple antiviral active components in the arithoke, as caffeoylquinic acid compounds (1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, 4-O-caffeoylquinic acids and 5-O-caffeoylquinic acids) and di-coffee mesitoyl quinine acid compounds (1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids) etc.
Coffee mesitoyl quinine acid compounds is a class polyphenolic compound that is formed by quinic acid and coffic acid condensation; extensively be present in the vegitabilia; the caffeoylquinic acid compounds 1-O-caffeoylquinic acids of Fen Buing widely; the 3-O-caffeoylquinic acids; 4-O-caffeoylquinic acids and 5-O-caffeoylquinic acids are isomers; the difference of this compounds is the link position difference of coffee acyl on quininic acid of a part, and molecular formula is C
16H
18O
9And two caffeoyl quinic acid compounds are 1; 3-two-O-caffeoylquinic acids; 1,5-two-O-caffeoylquinic acids, 3; 5-two-O-caffeoylquinic acids; 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids be isomers each other also; between five difference be the link position difference of two coffee acyls on quininic acid, molecular formula is C
25H
24O
12, molecular weight is 516.Structural formula is as follows:
R
1=coffee acyl, R
2=R
3=R
4=H 1-O-caffeoylquinic acids
R
2=coffee acyl, R
1=R
3=R
4=H 3-O-caffeoylquinic acids
R
3=coffee acyl, R
1=R
2=R
4=H 4-O-caffeoylquinic acids
R
4=coffee acyl, R
1=R
2=R
3=H 5-O-caffeoylquinic acids (chlorogenic acid)
R
1=R
2=coffee acyl, R
3=R
4=H 1,3-two-O-caffeoylquinic acids (cynarin)
R
1=R
4=coffee acyl, R
2=R
3=H 1,5-two-O-caffeoylquinic acids
R
1=R
4=coffee acyl, R
2=R
3=H 1,5-two-O-caffeoylquinic acids
R
2=R
4=coffee acyl, R
1=R
3=H 3,5-two-O-caffeoylquinic acids
R
2=R
3=coffee acyl, R
1=R
4=H 3,4-two-O-caffeoylquinic acids
R
3=R
4=coffee acyl, R
1=R
2=H 4,5-two-O-caffeoylquinic acids
Modern age, pharmacology test studies show that: coffee mesitoyl quinine acid compounds has multiple important physiologically active, mainly be at immune effect, suppress hepatitis B virus, virus of AIDS, hsv and influenza virus, suppress the synthetic and release of leukotriene, suppress histamine release, thereby can be used for antiviral, anti-inflammatory and treatment anaphylactic disease.Find that in addition it has potent antioxygenation, suppresses the anti-artery congee of lipoxygenase sclerization, platelet aggregation inhibitory activity and reducing blood lipid isoreactivity.
Coffee mesitoyl quinine acid compounds is the white powder solid, be soluble in hot water, hot methanol, hot ethanol, be insoluble to ether, the less organic solvent of benzene isopolarity, be heated and oxygenolysis take place or be converted into other di-coffee mesitoyl quinine acid compounds, in addition, three compounds are unstable in solution, can transform mutually, and this causes very big difficulty for their separation and purification.Therefore, set up a kind of efficiently, a plurality of coffee mesitoyl quinine acid compounds of separating and purifying high-purity from Chinese medicine or natural drug fast, have the modern Chinese herbal medicine preparation of special efficacy and make full use of the herb resource etc. of China significant to exploitation.
General coffee mesitoyl quinine acid compounds separating and extracting method is with methyl alcohol, ethanol or water refluxing extraction at present, concentrating under reduced pressure separation and Extraction liquid obtains medicinal extract, medicinal extract is suspended in water, successively with the solvent extraction of sherwood oil or normal hexane, chloroform or opposed polarities such as ethyl acetate, water saturated propyl carbinol.Coffee mesitoyl quinine acid compounds generally concentrates on the propyl carbinol phase, utilizes the purification on normal-phase silica gel column chromatography again, uses sherwood oil, ethyl acetate, chloroform, methyl alcohol equal solvent wash-out, and crosses Sephadex LH-20 column chromatography or preparative liquid chromatography obtains the simplification compound.These method steps complexity waste time and energy, and the rate of recovery is low, and the cost height is not suitable for industrialization.High speed adverse current chromatogram (high speed counter current chromatography, HSCCC) be a kind of liquid luquid partition chromatography technology that need not to use any solid-state supporting dielectric that grows up phase early 1980s, its ultimate principle is finally separated according to the partition ratio of separated material in the two-phase solvent system is different; This isolation technique is not owing to use solid support matrix, avoided the drawback that easily produces because of conventional fillers such as the sample loss that causes with solid packing generation irreversible adsorption, inactivation sex change, can make sample be able to whole recovery, and have that the separation capacity is big, performance by force, efficiently, characteristics fast, be particularly useful for the separation of preparation property, adverse current chromatogram has been widely used in the preparation separation and the purifying of field chemical substances such as biology, medicine, environmental protection.The development of countercurrent chromatography and application have solved the difficult problem that present coffee mesitoyl quinine acid compounds separating and purifying technology faces in conjunction with high performance preparative liquid chromatography separation and purification ability efficiently.
Summary of the invention
The technical problem to be solved in the present invention is the defective that overcomes traditional isolation technique, provide a kind of from crude extract separation and purification 1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, the 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4, the preparation method of 5-two-O-caffeoylquinic acids, this method has easy and simple to handle, fractional dose is big, rate of recovery advantages of higher.
Technical scheme of the present invention mainly comprises:
A) preparation of crude extract
The arithoke plant is pulverized, water-strong polar organic solvent extraction, extracting solution is evaporated to does not have the alcohol flavor, and behind centrifugal removal impurity, obtain clarifying concentrated solution, carry out macroporous adsorbent resin column chromatography, earlier closely colourless to be washed to, again with Different concentrations of alcohol aqueous solution gradient elution, receive 50～70% ethanol elution thing, elutriant is evaporated to nothing alcohol flavor, adds ethanol and measure 70～80%, remove precipitation to containing alcohol, get supernatant concentration, must contain the crude product of coffee mesitoyl quinine acid compounds.
B) separation and purification of target compound
After being rich in the crude extract vacuum-drying of coffee mesitoyl quinine acid compounds, separate preparation with high speed adverse current chromatogram in conjunction with high performance preparative liquid chromatography, collect the target component according to ultraviolet detection spectrogram or TLC collection of illustrative plates.
Target components after vacuum-drying, with the mixed solution of organic solvent or water-organic solvent carry out recrystallization get final product highly purified 1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids monomeric compound.
Among the preparation method, raw materials used preferred composite family arithoke in the step a); Extracting solvent is pure water mixed solvent, wherein alcohol can be methyl alcohol, ethanol, propyl alcohol, butanols, ethylene glycol etc. or their mixture, particular methanol or ethanol, the alcohol water mixed solvent can be the solvent of alcohol and water with any mixed, preferred alcohol and water mixed solvent, preferred therebetween 70～80% aqueous ethanolic solution.The weight of extracting solvent is 1～20 times of medicinal material weight, preferred 6～10 times; Extraction time is 0.5-5 hour, preferred 2 hours; Extraction time is 1-5 time, preferred 3 times; The column chromatography used medium is macroporous adsorbent resin, polymeric amide, reverse phase silica gel or dextrane gel class, preferred D101, AB-8, HPD100, HPD300, D1 or D2 type macroporous adsorbent resin, preferred 30～40% aqueous ethanolic solutions of eluting solvent; In the step b), the solvent system that preferred fat ester-Fatty Alcohol(C12-C14 and C12-C18)-water or halohydrocarbon-Fatty Alcohol(C12-C14 and C12-C18)-water is formed carries out high speed adverse current chromatogram, fatty ester can be an ethyl acetate, butylacetate, propyl acetate, ethyl formate, most preferably be ethyl acetate, halohydrocarbon can be a chloroform, methylene dichloride or tetrachloromethane, most preferably be chloroform, Fatty Alcohol(C12-C14 and C12-C18) can be a methyl alcohol, ethanol, Virahol or propyl carbinol, most preferably be methyl alcohol, wherein engine speed is just transferring 600～1200rpm to, preferred 800rpm, the flow velocity that moving phase pumps in the post is 0.5～5mL/min, preferred 1.5mL/min, the experiment condition temperature is 10～30 ℃, be preferably 25 ℃, on be stationary phase mutually, is moving phase mutually down; The solvent system that preferred fat alcohol-water-acid or fatty nitrile-water-acid is formed carries out high performance liquid chromatography and separates preparation, Fatty Alcohol(C12-C14 and C12-C18) can be methyl alcohol, ethanol etc., most preferably be methyl alcohol, fatty nitrile most preferably is acetonitrile, and acid can be formic acid, acetate, phosphate buffer soln etc., most preferably be acetate, wherein flow velocity is 5～20mL/min, preferred 10mL/min, and the experiment condition temperature is 10～30 ℃, be preferably 25 ℃, adopt the mode of gradient elution; Because the unstable of coffee mesitoyl quinine acid compounds, the target component that separates and collect, recrystallization after the vacuum-drying immediately, recrystallization solvent is the mixed solution of organic solvent or water-organic solvent, preferred 80% methanol aqueous solution or 80% aqueous ethanolic solution.
The present invention adopts high-speed counter-current chromatograph, it is finally separated according to the partition ratio of separated material in the two-phase solvent system is different, this isolation technique is not owing to use solid support matrix, avoided the drawback that easily produces because of conventional fillers such as the sample loss that causes with solid packing generation irreversible adsorption, inactivation sex change, can make sample be able to whole recovery, have that the separation capacity is big, performance by force, efficiently, characteristics fast.Purity not reached 98% caffeoyl quinic acid compounds separates in conjunction with high performance preparative liquid chromatography.By the control experiment condition, can obtain highly purified 1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids (purity is greater than 98%).The inventive method is suitable for the highly purified 1-O-caffeoylquinic acids of preparation from the crude extract of the coffee mesitoyl quinine acid compounds that contains different content of various technology approach preparations, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids monomeric compound, be suitable for various model high-speed counter-current chromatographs and high performance preparative liquid chromatography instrument and prepare the 1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, 4-O-caffeoylquinic acids, 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids monomeric compound can directly use a large amount of caffeoylquinic acids crude extracts or wherein several mixture or other similar syntheticss to carry out sample introduction.
Further specify the present invention below by embodiment and accompanying drawing.The following embodiment of mandatory declaration is used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention is simply improved the present invention and is all belonged to the scope of protection of present invention.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram (HPLC) of arithoke crude extract
Fig. 2 is a process flow sheet of the present invention
Embodiment
With the arithoke herb in 50 ℃ of dryings, pulverize.Get the 200g crude drug, put in the 2000mL round-bottomed flask, add 75% ethanol 1600mL, refluxing extraction 3 times, each 2h filters, merging filtrate, being evaporated to does not have the alcohol flavor, reclaims organic solvent.Concentrated solution is carried out the D101 macroporous adsorbent resin column chromatography, wash with water earlier to there not being the alcohol flavor, use 20% successively again, 40% aqueous ethanolic solution carries out wash-out, collect 40% ethanol eluate, being evaporated to does not have the alcohol flavor, adds ethanol to containing alcohol amount 80%, the elimination precipitation is got supernatant liquor vacuum-drying and must be contained the di-coffee mesitoyl quinine acid compounds crude product.Crude product is carried out the high speed adverse current chromatogram separation and purification, and condition is: rotating speed: 800rpm; Solvent system: ethyl acetate-n-butanol-water (3: 2: 5, v/v); Flow velocity: 1.5mL/min; Temperature: 25 ℃; On be stationary phase mutually, is moving phase mutually down; Stationary phase retention rate 65%.The operation steps of adverse current chromatogram is: be formulated in the separating funnel in proportion the HSCCC solvent system and thermal agitation, isolate phase up and down after the system branch balances each other, respectively ultrasonic degas 30min.Stationary phase is pumped into the spiral tube of high-speed counter-current chromatograph, treat that spiral tube is full of fully after, the opening speed controller makes high-speed counter-current chromatograph in the direction of the clock with the 800rpm rotation, simultaneously with flow pump people's moving phase of 1.5mL/min.After system reaches fluid dynamic equilibrium, sample is injected the separation pipeline by sampling valve.Effluent detects with UV-detector behind the post.Collect the chromatographic peak component according to color atlas, separation obtaining nine stream parts, and part merging of each stream is concentrated, and operational analysis type high performance liquid chromatograph carries out purity detecting.HPLC analysis condition: chromatographic column
C
18(100mm * 3.9mmi.d., 5 μ m), moving phase is methyl alcohol: 0.1% acetic acid (40: 60), flow velocity 0.8mL/min detects wavelength 326nm, 30 ℃ of column temperatures (with this understanding, arithoke crude extract high-efficient liquid phase chromatogram is seen Fig. 1).Less than stream part of 98% membrane filtration through 0.45 μ m, the colleges and universities' liquid chromatograph that enters the preparation type separates to purity, and preparative column is Zorbax ODS 9.4 * 250mm, and flow velocity is 10ml/min, obtains pure product.Each pure product carries out recrystallization with 80% ethanol and gets highly purified 1-O-caffeoylquinic acids, 3-O-caffeoylquinic acids, 4-O-caffeoylquinic acids, the 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids monomeric compound (process flow sheet is seen Fig. 2).
Claims (10)
1. high purity antiviral active components 1-O-caffeoylquinic acids in the arithoke, 3-O-caffeoylquinic acids, 4-O-caffeoylquinic acids, the 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4, the separation purification method of 5-two-O-caffeoylquinic acids is made up of preparation and separation and purification two portions of crude extract.It is characterized in that:
A) get contain the caffeoylquinic acid compounds and (or) plant of two coffee mesitoyl quinine acid compounds is raw material, pulverizes or be cut into segment, the mixed solution of water or water-strong polar organic solvent extracts, concentrated extracting solution reclaims solvent, concentrated solution;
B) get concentrated solution that step a) obtains through column chromatography, the mixed solvent wash-out of alcohol, ketone or they and water is used in washing then;
C) elutriant that step b) is obtained is evaporated to does not have the alcohol flavor or does not have the ketone flavor, dry must contain the caffeoylquinic acid compounds and (or) crude product of two coffee mesitoyl quinine acid compounds;
D) after the crude product alcohol precipitation removal of impurities that step c) is obtained, the use high speed adverse current chromatogram separates and purifying in conjunction with high performance preparative liquid chromatography, becomes shunting part according to detecting the spectrogram receiving target;
E) the stream part that step d) is obtained is evaporated to certain volume, and vacuum-drying gets target compound;
F) compound that step e) is obtained adopts the method for recrystallization to make with extra care, and obtains highly purified 1-O-caffeoylquinic acids, the 3-O-caffeoylquinic acids, 4-O-caffeoylquinic acids, 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4,5-two-O-caffeoylquinic acids monomeric compound.
2. high purity 1-O-caffeoylquinic acids according to claim 1, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, the 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4, the separation purification method of 5-two-O-caffeoylquinic acids, raw materials used preferred arithoke during its characterization step is a) also comprises the natural product that contains one or more compositions in above nine kinds of coffee mesitoyl quinine acid compounds; Extracting solvent is pure water mixed solvent, and the weight of extracting solvent is 1～20 times of medicinal material weight, and extraction time is 0.5～5 hour, and extraction time is 1～5 time; The column chromatography used medium is macroporous adsorbent resin, polymeric amide, reverse phase silica gel or dextrane gel class in the step b); Step c) is evaporated to not to be had the alcohol flavor or does not have the ketone flavor; The preferred alcohol precipitation process of removal of impurities in the step d), high speed adverse current chromatogram solvent systems preferred fat ester-Fatty Alcohol(C12-C14 and C12-C18)-water or halohydrocarbon-Fatty Alcohol(C12-C14 and C12-C18)-water carry out; High performance preparative liquid chromatography solvent systems preferred fat alcohol-water-acid or fatty nitrile-water-acid are carried out; Be vacuum-drying in the step e); Recrystallization solvent is the mixed solution of organic solvent or water-organic solvent in the step f).
3. according to the extraction plant of claim 1 and 2, wherein extract part is root, stem, leaf, seed, flower, skin, perhaps their mixture.
4. the preparation method of the plant milk extract of claim 3 with 70～80% extraction using alcohol, extracts 1～5 time in the step a), uses 3～10 times of amounts at every turn, extracts 0.5～4 hour at every turn.
5. according to the plant milk extract of claim 2, wherein the column chromatography used medium is the commercially available macroporous adsorbent resin or the macroporous adsorbent resin or the polyamide resin of polyamide resin and improvement in the step b), earlier with 0～15% aqueous ethanolic solution wash-out, follow aqueous ethanolic solution wash-out, collect 50～70% ethanol eluate with 50～70%.
6. according to the preparation method of claim 5, after the ethanol eluate of step c) is concentrated, add ethanol earlier, remove precipitation, get supernatant concentration, get the coffee mesitoyl quinine acid compounds crude product to containing alcohol amount 70～80%.
7. the total content of coffee mesitoyl quinine acid compounds is 10%-90% in the caffeoylquinic acids crude extract of one of claim 1～6.
8. according to the 1-O-caffeoylquinic acids described in the claim 2, the 3-O-caffeoylquinic acids, the 4-O-caffeoylquinic acids, the 5-O-caffeoylquinic acids, 1,3-two-O-caffeoylquinic acids, 1,5-two-O-caffeoylquinic acids, 3,5-two-O-caffeoylquinic acids, 3,4-two-O-caffeoylquinic acids and 4, the method for preparing purified of 5-two-O-caffeoylquinic acids, it is characterized in that the solvent system that preferred fat ester-Fatty Alcohol(C12-C14 and C12-C18)-water or halohydrocarbon-Fatty Alcohol(C12-C14 and C12-C18) in the described step d)-water is formed carries out high speed adverse current chromatogram, fatty ester can be an ethyl acetate, butylacetate, propyl acetate, ethyl formate etc.; Halohydrocarbon can be chloroform, methylene dichloride or tetrachloromethane etc., and Fatty Alcohol(C12-C14 and C12-C18) can be methyl alcohol, ethanol, Virahol or propyl carbinol.In chloroform-methanol-aqueous systems, the preferable amount volume ratio is 2: 2: 1; In ethyl acetate-n-butanol-water system, the preferable amount volume ratio is 3: 2: 5, more than be stationary phase mutually, is moving phase mutually down.The solvent system that preferred fat alcohol-water-acid or fatty nitrile-water in the described step d)-acid is formed carries out high performance preparative liquid chromatography to be separated, Fatty Alcohol(C12-C14 and C12-C18) can be methyl alcohol and ethanol etc., fatty nitrile can be an acetonitrile etc., and acid can be acetate, formic acid, phosphate solution etc., adopts gradient elution.
9. the preparation method of a kind of high purity coffee mesitoyl quinine acid compounds according to claim 8, it is characterized in that: in high-speed counter-current chromatograph, earlier be filled with the counter current chromatograph pillar with stationary phase, regulate engine speed, moving phase is pumped in the post, after treating that whole system is set up running balance, by the sampling valve sample introduction, according to detector uv-spectrogram receiving target composition; Wherein engine speed is just transferring 600～1200rpm to, and the flow velocity that described moving phase pumps in the post is 0.5～5mL/min, and the experiment condition temperature is 10～30 ℃; In the high performance preparative liquid chromatography instrument, chromatographic column is half preparation type C18 post, and flow velocity is 5～20mL/min, and the experiment condition temperature is 10～30 ℃.
10. the purification process of nine target products according to claim 2 is characterized in that, in the described step f), preferred 80% methyl alcohol or 80% ethanol carry out recrystallization.
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